Detection of Mycobacterium tuberculosis in blood by use of the Xpert MTB/RIF assay

Abstract

We have developed a novel blood lysis-centrifugation approach for highly sensitive Mycobacterium tuberculosis detection in large volumes of blood with the Xpert MTB/RIF assay. One through 20 ml of blood was spiked with 0.25 to 10 CFU/ml of the M. tuberculosis surrogate M. bovis BCG. Multiple replicates of each sample were processed by a new lysis-centrifugation method and tested with the Xpert MTB/RIF assay. The assay was very sensitive with increased blood volumes. In the 20-ml samples, BCG was detected in blood spiked with 10, 5, 1, and 0.25 CFU/ml 100, 100, 83, and 57% of the time, respectively, compared to 100, 66, 18, and 18%, of the time, respectively, in 1-ml blood samples. Assay sensitivity was influenced by the type of anticoagulant used, with acid-citrate-dextrose solution B (ACD-B) providing the best results. A limit of detection of 10 CFU/ml was established with BCG spiked into ACD-B-treated blood, and 92, 36, and 33% of the samples with 5, 1, and 0.5 CFU/ml, respectively, were assay positive. The lysis buffer was stable both at room temperature and at 4°C for 2 months. The assay was tested with blood stored for 8 days without a change in sensitivity as measured by cycle threshold. This new assay format extends the capability of the Xpert MTB/RIF test, enabling up to 20 ml of blood to be tested rapidly for the presence of M. tuberculosis. This approach may be a useful method to detect extrapulmonary tuberculosis and the risk of death in immunocompromised patients.

Fig 1

Flow chart describing our large-volume blood sample-processing methodology.

Fig 2

Effect of blood volume on the probability of mycobacterium detection. Various numbers of BCG CFU per milliliter were spiked into different volumes of blood. The percentages of samples among seven samples per concentration that were positive at each concentration are shown.

Fig 3

Comparison of various anticoagulants. Blood samples anticoagulated with ACD-A, ACD-B, or EDTA were spiked with 5 and 10 CFU/ml of BCG in 5 ml whole blood. The proportion of samples that were positive (bars) and the C_T value of the rpoB assay (■) are shown for each condition. The number of replicates run with each anticoagulant type was 4 to 7.

Fig 4

LOD of mycobacteria in ACD-B-anticoagulated blood. BCG was spiked into 5-ml blood aliquots. The proportion of positive samples for each CFU concentration out of 15 samples tested per concentration is shown. Each replicate is indicative of a blood sample from one healthy individual.

Fig 5

Solution and sample stability. Stability studies were performed by running Xpert MTB/RIF assays with lysis solution stored for 0 to 90 days at RT (♢) or under refrigerated conditions (4 to 8°C) (■) (A) and BCG-spiked blood samples stored for 0 to 8 days (B). Each time point shows the mean of five test results. An increase in the C_T (not shown) would have indicated that a stability storage limit had been reached.